The present invention relates to targeted polymerized liposomes for oral and/or mucosal delivery of vaccines, allergens and therapeutics. In particular, the present invention relates to polymerized liposomes which have been modified on their surface to contain a molecule or ligand which targets the polymerized liposome to a specific site or cell type in order to optimize the immune response to the encapsulated antigen or the efficacy of the encapsulated therapeutic. More particularly, the present invention relates to the use of polymerized liposomes modified to contain a carbohydrate or lectin on their surface to deliver vaccines to mucosal epithelium. The present invention further relates to the synthesis, preparation and use of the modified polymerized liposomes of the present invention as, or in, pharmaceutical compositions for oral delivery of drugs and vaccines.
Drug Delivery
Drug delivery takes a variety of forms, depending on the agent to be delivered and the administration route. The most convenient way to administer drugs into the body is by oral administration. However, many drugs, in particular proteins and peptides, are poorly absorbed and unstable during passage through gastrointestinal (G-I) tract. The administration of these drugs is generally performed through parenteral injection.
Although oral vaccination is more convenient, vaccines are generally given through injection. This is particularly true with killed or peptidic vaccines, because of their low absorbability and instability in the G-I tract. A problem with systemic immunization is that it may not effectively induce mucosal immune responses, particularly production of IgA, that are important as the first defense barrier to invaded microorganisms. For this reason, it would be beneficial to provide oral vaccination, if the problems of low absorbability and instability could be overcome.
Controlled release systems for drug delivery are often designed to administer drugs to specific areas of the body. In the gastrointestinal tract it is important that the drug not be eliminated before it has had a chance to exert a localized effect or to pass into the bloodstream.
Enteric coated formulations have been widely used for many years to protect drugs administered orally, as well as to delay release several microsphere formulations have been proposed as a means for oral drug delivery. For example, PCT/US90/0643 and PCT/US90/06433 by Enzytech discloses the use of a hydrophobic protein, such as zein, to form microparticles; U.S. Pat. No. 4,976,968 to Steiner et al. discloses the use of xe2x80x9cproteinoidsxe2x80x9d to form microparticles; and European Patent Application 0,333,523 by the UAB Research Foundation and Southern Research Institute discloses the use of synthetic polymers such as polylactide acid-glycolic acid to form in microspheres.
Particles less than ten microns in diameter, such as the microparticles of EPA 0,333,523, can be taken up by cells in specialized areas, such as Peyer""s patches and other intestinal mucosal lymphoid aggregates, located in the intestine, especially in the ileum, into the lymphatic circulation. Entrapping a drug or antigen in a microparticulate system can protect the drug or antigen from acidic and enzymatic degradation, yet still allow the drug or antigen to be administered orally, where they are taken up by the specialized uptake systems, and release the entrapped material in a sustained manner or are processed by phagocytic cells such as macrophages. When the entrapped material is a drug, elimination of the first-pass effect (metabolism by the liver) is highly advantageous.
Liposomes
Liposomes have been proposed for use as an oral drug delivery system, for example, by Patel and Ryman, FEBS Letters 62(1), 60-63 (1976). Liposomes are typically less than 10 microns in diameter, and, if they were stable to passage through the G-I tract, may be absorbed through Peyer""s patches. Liposomes also have some features that should be advantageous for a particulate system for oral drug or antigen delivery. The phospholipid bilayer membrane of liposomes separates and protects entrapped materials in the inner aqueous core from the outside. Both water-soluble and -insoluble substances can be entrapped in different compartments, the aqueous core and bilayer membrane, respectively, of the same liposome. Chemical and physical interaction of these substances can be eliminated because the substances are in these different compartments. Further, liposomes are easy to prepare. However, liposomes are physically and chemically unstable, and rapidly leak entrapped material and degrade the vesicle structure. Without fortifying the liposomes, they are not good candidates for oral drug or antigen delivery.
Several methods have been tried to fortify liposomes. Some methods involved intercalating cholesterol into the bilayer membrane or generating the liposomes in the presence of polysaccharides. These methods are not useful in making liposome for oral delivery since during oral delivery liposomes are exposed to an acidic pH in the stomach and bile salts and phospholipases in intestine. These conditions break down the cholesterol and polysaccharide in the liposomes.
Investigators have explored the improved stability of polymerized liposomes, however in the area of drug delivery their ultimate utility remains uncertain (Regen, 1987 in Liposomes From Biophysics to Therapeutics, edit. Ostro, Marcel Dekker, N.Y.). Polymerized liposomes have been developed in attempts to improve oral delivery of encapsulated drugs (Chen et al. WO 9503035). The ability of liposomes derivatized with wheat germ agglutinin to better survive the G-I tract has also been investigated (Chen et al., 1995, Proceed. Internat. Symp. Control. Rel. Bioact. Mater. 22; Chen et al., 1995 Proc. 3rd U.S. Japan Symposium on Drug Delivery).
However, to the inventors"" knowledge, to date the utility of conventional liposomes for oral delivery is still questionable. Similarly, whether polymerized liposomes are more advantageous than conventional or unpolymerized liposomes for oral drug delivery is still unclear since improved stability alone may not be sufficient for oral drug delivery, particularly oral vaccination. Thus, there remains a need for drug and antigen delivery devices that can survive the harsh conditions in the G-I tract, and effectively deliver the drug, antigen or any other therapeutic.
The present invention encompasses polymerized liposomes which have been modified, preferably on their surface, to contain a molecule or ligand which targets the polymerized liposome to a specific site. The invention also encompasses the use of the modified polymerized liposomes for the oral delivery of drugs and antigen delivery systems. In particular, the polymerized liposomes of the present invention are modified to contain a carbohydrate moiety or lectin which targets the liposomes to Peyer""s Patch cells and mucosal epithelium.
The present invention is based on, inter alia, Applicants"" discovery that the polymerized liposomes of the present invention have surprisingly enhanced stability against the harsh environment of the gastrointestinal tract particularly when compared to unpolymerized liposomes. Further, the modification of the polymerized liposomes to include lectins which have a binding affinity for the mucosal cells of the Peyer""s Patch resulted in unexpectedly enhanced and rapid uptake of intact liposomes. For example, uptake or absorption into mucosal tissue is enhanced by the polymerized liposomes of the present invention whether administered orally, intranasally, sublingually, buccally or rectally. That the modified polymerized liposomes of the present invention are an optimal system for the oral and/or mucosal delivery of vaccines, allergens, drugs and therapeutics is demonstrated by the working examples described infra.
The present invention relates to the modification of the polymerized liposomes to contain molecules or ligands which target the liposomes to a specific site in order to optimize uptake of the liposome and its encapsulated therapeutic, and/or to optimize the immune response of the encapsulated antigen or the efficacy of the encapsulated drug. The polymerized liposomes of the present invention can be modified by a variety of moieties and molecules, including but not limited to, glycoproteins, carbohydrates, lectins, antibodies, antibody fragments and other molecules or proteins that may be used to target mucosal epithelium. In a preferred embodiment of the present invention, the surface of the polymerized liposomes are modified to contain carbohydrate moieties or lectins which have affinity for mucosal epithelium cells and Peyer""s Patch cells. In yet another embodiment of the invention, the surfaces of the polymerized liposomes are modified to contain monoclonal antibodies which have affinity for a specific cell-surface protein. The modified polymerized liposomes are than targeted to a specific cell or organ as intact particles.
The modified polymerized liposomes of the present invention may be utilized for the delivery of a wide variety of compounds, allergens and antigens, including, but not limited to insulin peptides, diphtheria toxin antigens and influenza antigens. The modified polymerized liposomes of the present invention have particular utility in the oral and/or mucosal delivery of vaccines and antigen release devises. The modified polymerized liposomes of the present invention may also be utilized for the oral delivery of a wide variety of therapeutics, including but not limited to, chemotherapy agents for the treatment of cancer; cytokines, including interferon; and hormones including insulin, human growth hormone (HGH), fertility drugs, calcitonin, calcitriol and other bioactive steroids.
The present invention relates to the synthesis, preparation and use of the modified polymerized liposomes. The liposomes of the present invention are composed of phospholipids which are polymerized by covalent bonding to each other. Covalently bonding the layers adds strength, resulting in a less fluid unpolymerized liposome. The less fluid bi-layer membrane suppresses leakage. Further, the detergent-like bile salts in the intestine cannot solvate the phospholipid molecules. These cross-linked membranes are strong enough to maintain their structure even if the phospholipids undergo hydrolysis at low pH and enzymatic degradation by phospholipases. Thus, polymerized liposomes reach the ileum of the G-I tract as intact particulates, and are absorbed. In addition, the ligand target of the polymerized liposome must be stable to the harsh conditions of the G-I trace and the link or bond to the polymerized liposome must be sufficiently stable to remain intact until the liposome is targeted or delivered.
Definitions
As used herein; the term xe2x80x9cliposomexe2x80x9d is defined as an aqueous compartment enclosed by a lipid bilayer. (Stryer, Biochemistry, 2d Edition, W. H. Freeman and Co., p. 213 (1981)). The liposomes can be prepared by a thin film hydration technique followed by a few freeze-thaw cycles. Liposomal suspensions can also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, incorporated herein by references in its entirety.
As used herein, the term xe2x80x9cpolymerized liposomexe2x80x9d is defined as a liposome in which the constituent phospholipids are covalently bonded to each other by inter and intra molecular interactions. The phospholipids can be bound together within a single layer of the phospholipid bilayer (the leaflets) and/or bound together between the two layers of the bilayer.
The degree of crosslinking in the polymerized liposomes can range from 30 to 100 percent, i.e., up to 100 percent of the available bonds are made. The size range of polymerized liposomes is between approximately 15 nm to 10 xcexcm. The polymerized liposomes can be loaded with up to 100% of the material to be delivered, when the material is hydrophobic and attracted by the phospholipid layers. In general, about 5 to about 40 percent of the material is encapsulated when the material is hydrophilic.
As used herein, the term xe2x80x9ctrap ratioxe2x80x9d is defined as the ratio of inner aqueous phase volume to total aqueous phase volume used.
As used herein, the term xe2x80x9cradical initiatorxe2x80x9d is defined as a chemical which initiates free-radical polymerization.
As used herein, the term xe2x80x9creverse phase evaporation techniquexe2x80x9d is defined as a method involving dissolving a lipid in an organic solvent, adding a buffer solution, and evaporating the organic solvent at reduced pressure, as described by Skoza, F. Jr., and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA, Volume 75, No. 9, pp. 4194-4198 (1978).
As used herein, the term xe2x80x9cfreeze-thaw technique,xe2x80x9d or xe2x80x9cF-T,xe2x80x9d is defined as freezing a suspension in liquid nitrogen, and subsequently thawing the suspension in a roughly 30xc2x0 C. water bath.
As used herein, the terms xe2x80x9cmucosaxe2x80x9d or xe2x80x9cmucosalxe2x80x9d refers to a mucous tissue such as epithelium, lamina, propria, and a layer of smooth muscle in the digestive tract. Mucosal delivery as used herein is meant to include delivery through bronchi, gingival, lingual, nasal, oral, and intestinal mucosal tissue.
As used herein, the term xe2x80x9cbuffer solutionxe2x80x9d is defined as an aqueous solution or aqueous solution containing less than 25% of a miscible organic solvent, in which a buffer has been added to control the pH of the solution. Examples of suitable buffers include but are not limited to PBS (phosphate buffered saline), TRIS (tris-(hydroxymethyl)aminomethane), HEPES (hydroxyethylpiperidine ethane sulfonic acid), and TES 2-[(tris-hydroxymethyl)methyl amino-1-ethanesulfonic acid.
As used herein, the term xe2x80x9cleafletsxe2x80x9d is defined as a single layer of phospholipid in the bilayer forming the liposome.